FOXP3 and CXCR4-positive regulatory T cells in the tumor stroma as indicators of tumor immunity in the conjunctival squamous cell carcinoma microenvironment

Conjunctival squamous cell carcinoma (SCC) is the most common ocular surface neoplasia. The purpose of this retrospective study was to examine the role of regulatory T cell (Treg) activity in tumor immunity and investigate the tumor microenvironment as a new treatment focus in conjunctival SCC. Cancer progression gene array and immunohistochemical analyses of FOXP3 as a Treg marker, CD8 as a tumor-infiltrating lymphocyte marker, and CXCR4 expression on activated Tregs were conducted in a series of 31 conjunctival SCC cases. The objective was to investigate the immunoreactive response in tumor cells and stromal cells in the cancer microenvironment. The stroma ratio in tumor cells was investigated by monitoring α-smooth muscle actine (SMA) expression between carcinoma in situ (Tis) and advanced carcinoma (Tadv) (P<0.01). No significant change in PD-L1 expression was observed in this study (P = 0.15). Staining patterns of FOXP3, CD8, and CXCR4 were examined separately between tumor cells and stromal cells in SCC tumors. Differences in staining of FOXP3 in Tregs and CD8 in tumor-infiltrating lymphocytes in tumor stroma in the Tis group were observed compared with the Tadv group (each P<0.01). In addition, double immunostaining of CXCR4/FOXP3 was correlated with progression-free survival (P = 0.049). Double immunostaining of CXCR4/FOXP3 correlated with American Joint Committee on Cancer T-stage, independent of age or Ki67 index (P<0.01). Our results show that FOXP3 and the CXCR4/FOXP3 axis are important pathologic and prognostic factors of ocular surface neoplasia, including SCC. The tumor microenvironment of conjunctival SCC should be considered in the future development of treatment options.

Introduction Ocular surface squamous neoplasia (OSSN) includes several diseases, such as conjunctival premalignant dysplasia, carcinoma in situ (Tis), and advanced conjunctival squamous cell carcinoma (Tadv) [1]. The annual incidence of OSSN in the United Kingdom is 0.53 cases/million population/year (conjunctival intraepithelial neoplasia: 0.43 cases/million population/year; SCC: 0.08 cases/million population/year) [2,3]. In the United States, the incidence of SCC is 5-fold higher among males and whites [4]. However, no large-scale epidemiologic studies have been performed in populations in the Far East. In addition, recent studies have reported associations between findings such as loss of retinoblastoma protein/P16/cyclin D1, strong p53 immunostaining, and ADAM3A overexpression and the molecular pathology of SCC [5][6][7]. In recent years, research on factors related to tumor immunity, including programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) has advanced worldwide to encompass aspects ranging from basic research to clinical indications in head-neck SCC [8][9][10][11]. In skin SCC, the efficacy of Pembrolizumab and Cemiplimab and the potential for first-line treatment have been reported [12,13]. Recent ophthalmologic-related research demonstrated that PD-L1 is expressed in almost half of conjunctival SCC cases and indicated the potential application of immune checkpoint blockade as a treatment strategy for conjunctival SCC [6,14]. Other research indicates that a significant number of sebaceous carcinoma cases involve expression of PD-L1 at therapeutic levels with tumor-infiltrating T cells.
Regulatory T cells (Tregs) play a major role in tumor immunity in the tumor microenvironment [15][16][17][18][19]. In particular, CD4(+) Tregs expressing the transcription factor forkhead box P3 (FOXP3) are abundant in tumor tissues, where they appear to hinder the induction of effective antitumor immunity [15]. Tregs function in maintaining immunologic self-tolerance by actively suppressing self-reactive lymphocytes, and the gene encoding FOXP3 is a key regulator of Treg development [20]. Freeman et al. reported that a high CD4+ T cell to CD8+ T cell ratio may be an immunologic diagnostic indicator of late-stage dermal SCC development in immune-competent patients [21]. Considerable research attention has focused on the relationship between the role of local tumor-infiltrating lymphocytes (TILs [CD4+ and/or CD8+ T cells]) associated with FOXP3+ Tregs in the tumor microenvironment and the prognosis of SCC patients. Elucidating details regarding the interactions between Tregs in the tumor microenvironment and other molecules is another important area of research. The present study investigated the expression of FOXP3 in tumor tissues from SCC patients in order to assess the molecular association between Tregs and prognosis in East Asian patients with conjunctival SCC.

Selection of cases and collation of clinicopathologic data
This study was approved by the Institutional Review Boards of Osaka City University and Kobe Kaisei Hospital and adhered to the tenets of the 1964 Declaration of Helsinki. Written informed consent was obtained from all patients before enrollment. We identified 29 patients treated by ophthalmologists (AA, MT) between November 2007 and April 2020 from whom we were able to procure tissue blocks with residual tumors. Demographic information (age at initial diagnosis and at presentation to our institution; sex) and primary tumor features (disease status at presentation [primary or recurrent] and in situ versus invasive disease) were obtained from each patient enrolled. The American Joint Committee on Cancer (AJCC) stage, local recurrence (anatomic site and date), metastases (regional or distant, and date), vital status at last follow-up, cause of death if deceased, type of surgery, and adjuvant therapy were also recorded.

Image analysis
Tissues immunostained for FOXP3, CD8, α-SMA, PD-L1, and CXCR4 were evaluated in a blinded manner by two specialists (MT and AK). The first field for evaluation in each tumor lesion was selected randomly, and subsequently, 10 fields were examined systematically at 400× magnification using a mesh.
In order to investigate localization in greater detail, the FOXP3 and CD8 staining patterns in tissues were examined separately for tumor (TM) and tumor stroma (ST). For FOXP3/ CXCR4 double staining, the number of Tregs co-expressing FOXP3/CXCR4 was determined from four (×40) high-power fields of lymphoid-rich infiltrate within the tumor, according to previously reported methods [22]. Samples were semi-quantitatively divided into groups based on a FOXP3/CXCR4 double staining score in the range 0 to 3 (0, none: 0:1 field; 1, weak: 1-2> 1 field; 2, strong: 3> 1 field) according to the presence or absence and intensity of staining.

Immunofluorescence
The FFPE sections (3 μm) were deparaffinized for fluorescence immunohistochemistry. For heat-induced antigen retrieval, tissue sections were immersed in 0.01 mol/L citrate buffer and treated in a microwave oven at 620 W. The sections were then blocked with 1% bovine serum albumin for 1 h at room temperature. Sections were then incubated with the primary antibody, anti-FOXP3 (1:50) or anti-CXCR4 (1:50), overnight at 4˚C, washed with PBS, and incubated with donkey anti-rabbit-Alexa Fluor 488 (1:1000; Thermo Fisher Scientific, Tokyo, Japan), goat anti-mouse-Alexa Fluor 594 (1:1000, #ab150073, Abcam) for 30 min at room temperature and placed on slides using mounting medium with DAPI (Vectashield antifade medium with DAPI: Vector Laboratories).

Gene expression in tumors
The expression of major cancer-associated genes in tumor tissues was compared between patients with Tis and Tadv using NanoString analysis.
Genes examined included EGFR, HIF1A, ICAM1, IL-6, CXCR-4, MAPK1, MMP2, MMP9, NFKB1, NOTCH1, ROCK1, ROCK2, TGFB1, TNF, VEGFA, and WNT5A. Archival formalinfixed paraffin-embedded tumor tissues were retrieved and manually macrodissected. Total mRNA was isolated using a Qiagen miRNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Isolated RNA was quantified using a NanoDrop system (Thermo Scientific, Wilmington, DE, USA) and regarded as adequate if a sample contained a minimum of 400 ng. The samples were subsequently analyzed using an nCounter PanCancer Progression Panel (NanoString, Seattle, WA, USA) according to the manufacturer's instructions. NanoString data were processed using the R statistical programming environment (v3.4.2). Considering counts obtained for positive control probe sets, raw NanoString counts for each gene were subjected to technical factorial normalization, which was carried out by subtracting the mean count plus two standard deviations from the CodeSet inherent negative controls. Subsequently, biological normalization using the included mRNA reference genes was performed. Additionally, all counts with P>0.05 by one-sided t-test versus negative controls plus two standard deviations were interpreted as not expressed over basal noise.

Statistical analyses
Clinical and histopathologic characteristics were summarized using descriptive statistics. Correlations between immunohistochemical, demographic, and clinicopathologic factor data were assessed using the Wilcoxon rank sum and Fisher's exact tests. Analysis of covariance was used to analyze the relationship between T-stage of tumors and FOXP3/CXCR4-positive Tregs. With respect to regression analyses, an explanatory variable that roughly divided the number of cases by 15 was considered appropriate [23]. Progression-free survival (PFS) was defined as the time from surgery to disease recurrence or death from any cause. Cox regression modeling was used to evaluate correlations between clinicopathologic and immunohistochemical features and survival outcomes. Statistical analyses were performed using SPSS Statistics software, version 22 (IBM Japan, Tokyo, Japan). P<0.05 was considered indicative of statistical significance.

Results
Clinicopathologic findings of our cohort are summarized in Table 1. All 31 patients (100%) were East Asian and included 17 men and 14 women, with a mean age at presentation of 77.9 years. Sixteen patients (51%) had invasive SCC, and 15 (49%) had an in situ tumor. Primary orbital exenteration was necessary for local disease control in three patients (9%), and two patients (6%) underwent additional orbital exenteration. Nine patients (29%) underwent adjuvant therapy, most commonly additional local surgery. Topical chemotherapy and radiation therapy were performed in one patient in the adjuvant therapy group. One patient in the adjuvant therapy group died with disease 11 months after diagnosis of regional and lung metastases; another patient was alive without disease at 50 months after diagnosis of regional  Table 2).
In the evaluation of the stroma ratio in tumor tissue based on α-SMA expression, scores were 1.13±0.35 in the Tis group and 1.81±0.83 in the Tadv group (P<0.01) (Fig 2). There was no significant change in PD-L1 expression in our study (P = 0.15).

Discussion
To the best of our knowledge, this is one of the first studies to investigate immunity in the tumor microenvironment in conjunctival SCC and evaluate the prognostic significance of TILs expressing FOXP3, CD8, and CXCR4/FOXP3 comparing the Tis and Tadv groups.
In addition, evaluation of the stroma using α-SMA and the expression of PD-L1 were also investigated in SCC and have been previously reported in other regions [25,26].
In this study, we found a clear difference in expression of FOXP3 between the Tis and Tadv groups in TM and ST. Expression of CD8 was also significantly different in the ST group. Stromal ratios in tumor cells based on α-SMA expression differed significantly between the Tis and Tadv groups. α-SMA expression was high in the stroma of tumor tissue of patients with advanced stage. However, there was no significant difference in PD-L1 expression in our study.
In addition, CXCR4/FOXP3 double staining in Treg was carried out to confirm the TIL balance in the tumor microenvironment. There was a significant difference between the Tis and Tadv groups. Increased expression of CXCR4 in the Tadv group was also confirmed by both immunohistochemical analysis and our previous analysis of mRNA expression [24].
Recent research has shown that immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome are caused by mutations in FOXP3, indicating that this is the master gene of Tregs [20]. The primary mediator cells in the tumor microenvironment are Tregs, and many studies have investigated FOXP3 expression and the CD8 or other lymphocyte marker ratios in TILs as Treg markers [15,22]. In ophthalmologic research, some studies have reported that Tregs play a role in protecting tissues from autoimmune diseases, such as dry eye or graft versus host disease after bone marrow transplantation, as well as ocular malignant neoplasms [27,28]. However, some studies in the field of systemic oncology have reported that Tregs block TILs in the tumor microenvironment, thus promoting tumor progression. Therefore, local suppression of Tregs in the tumor is considered a new axis of targeted treatment, and a number of studies examining this possibility are underway [17][18][19].
Our study is the first to examine the expression of FOXP3, CD8, and CXCR4/FOXP3 in conjunction with tumor progression and prognosis in patients with conjunctival SCC. This is a new finding regarding tumor escape from immunity, which was also shown in our case of conjunctival SCC. In addition, double immunostaining of CXCR4 and FOXP3 in conjunctival SCC indicated that these factors may play cooperative molecular roles. When a tumor progresses, the ratio of FOXP3 to CD8 expression shifts toward FOXP3. This is consistent with previous studies of SCC tumors in other areas and suggests that the change in the FOXP3/CD8 ratio might affect tumor growth by enabling tumor cells to evade attack by TILs in the tumor microenvironment [8][9][10]. In addition. our results also show that double staining of CXCR4 / FOXP3 is significantly associated with PFS in the COX hazard model. However, due to the low number of cases and the low incidence of events, multivariate analysis has not been performed and confounding factors cannot be ruled out.
In order to investigate the mechanism by which tumor cells induce Tregs, we comprehensively examined the expression of genes thought to play a major role in tumor growth. We found that inflammation-and ischemia-related factors such as IL-6 and HIF-1α were overexpressed. Previous studies have reported that these factors induce Tregs to proliferate and aggregate into tumors [29][30][31]. Furthermore, vascular endothelial growth factor (VEGF)-associated ischemia has been shown to inhibit dendritic cell maturation via the NF-κB pathway, which would diminish the presentation of cancer cell antigens and promote the growth of cancer cells in the ocular microenvironment [32]. Indeed, in our study, expression of NF-κB was significantly lower in the Tadv group than the Tis group, although this was evaluated only at the mRNA level.
Among the genes examined, we were very interested in the expression of CXCR4, as CXCR4 is reportedly associated with tumor growth, and some studies indicated that CXCR4 affects the proliferation and activity of Tregs in the tumor microenvironment [22,33,34]. In addition, a relationship between CXCR4 and FOXP3-positive Tregs has been reported [22]. The results of our study also suggest that IL-6 and HIF-1α are associated with CXCR4 and that CXCR4 expression may be associated with an increase in FOXP3-positive Tregs. This means that inflammation is the anti-tumor immune effect of CXCR4-axis Tregs due to ischemia, and to our knowledge, ours is the first report of this association in the area of conjunctival SCC (Fig 4).
These findings have important implications in SCC, which is strongly resistant to targeted therapies but could potentially respond to immunotherapies that target Tregs. Furthermore, it is possible that Treg recruitment by CXCR4 in these cancers could be modulated by treatment directed against hypoxia pathway factors, including HIF-1α (Fig 4). Thus, the development of treatment strategies based on reagents directed against Tregs, antibodies that block CXCR4, or inhibitors of HIF-1α and VEGF represents a potentially fruitful area of research.
This study has important limitations. First, regarding FOXP3 and CXCR4 expression in OSSN, changes associated with benign disease and age-related changes in normal tissues may not have been sufficiently investigated. Further studies, including multi-institutional studies and an increase in the number of cases, will be needed in the future.
In gene expression array, the methods was macro-dissection, therefore, in some cases, there might be no distinction between tumor tissue and stroma. For that reason, this difference in genetic expression may be a combination of the tumor and the stroma around the tumor.
In addition, the size of our study cohort was small (N = 31), and the length of follow-up (less than 1 year in some patients) may not have been sufficient for long-term outcome analyses. Therefore, additional studies will be needed to corroborate our findings.
In conclusion, the results of this study indicate that FOXP3 and CXCR4, which are indicators of Treg activity, are potential molecular targets and prognostic factors in the treatment of OSSNs, including SCC. Our findings also suggest that the tumor microenvironment of conjunctival SCC must be considered in the future development of treatment options. Supporting information S1 Data. (XLSX)